OCL 2017, 24(4), D407 © F. Hubert et al., published by EDP Sciences, 2017 DOI: 10.1051/ocl/2017023 OCL Oilseeds & fats Crops and Lipids Topical issue on: Available online at: LIPIDES DU FUTUR www.ocl-journal.org LIPIDS OF THE FUTURE PROCEEDINGS Lipids and lipolytic enzymes of the microalga Isochrysis galbana Florence Hubert, Laurent Poisson*, Céline Loiseau, Laurent Gauvry, Gaëlle Pencréac’h, Josiane Hérault and Françoise Ergan Laboratoire Mer, Molécules, Santé (EA 2160), Université du Maine, IUT de Laval, 52 rue des Drs Calmette et Guérin, BP 2045, 53020 Laval cedex, France Received 27 January 2017 – Accepted 21 April 2017 Topical Issue Abstract – Marine microalgae are now well-known for their ability to produce omega-3 long chain polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Among these microalgae, Isochrysis galbana has received increasing interest especially because of its high DHA content and its common use in hatchery to feed fish larvae and clams. Moreover, lipolysis occurring from the biomass harvest stage suggests that I. galbana may contain lipolytic enzymes with potential interesting selectivities. For these reasons, the potential of this microalga for the production of valuable lipids and lipolytic enzymes was investigated. Lipid analysis revealed that DHA is mainly located at the sn-2 position of the phospholipids. Thus, I. galbana was considered as an interesting starting material for the lipase catalyzed production of 1-lyso-2-DHA-phospholipids which are considered as convenient vehicles for the conveyance of DHA to the brain. Lipids from I. galbana can also be used for the enzyme- catalyzed production of structured phospholipids containing one DHA and one medium chain fatty acid in order to combine interesting therapeutic and biological benefits. Starting from total RNA extract from I. galbana, coding sequences of putative lipolytic enzymes were obtained by RACE and Nested PCR. The heterologous expression of a sequence designated IgTeCe was implemented. An expression plasmid was constructed by ligating the coding sequence to a plasmid vector and then cloned and expressed in E. coli. Results showed the effective functionality of plasmid construction for the production of a recombinant protein with the expected molecular mass. Moreover, local alignment using BLASTP and biochemical evidences support the hypothesis that the expressed protein is a thioesterase. Keywords: microalgae / v-3 polyunsaturated fatty acids / phospholipids / lipolytic enzymes Résumé – Lipides et enzymes lipolytiques de la microalgue Isochrysis galbana. Les microalgues marines sont maintenant bien connues pour leur aptitude à produire des acides gras à longue chaîne de la série v-3 comme l’acide docosahexahénoïque (DHA) ou eicosapentaénoïque (EPA). Parmi ces microalgues, Isochrysis galbana connaît, ces dernières années, un intérêt croissant du fait d’un contenu lipidique riche en DHA et de son importante utilisation en écloserie pour nourrir les larves de poissons et les bivalves. D’autre part, la lipolyse souvent observée dès l’étape de collecte de la biomasse ainsi que la forte teneur en DHA suggère qu’I. galbana pourrait contenir des enzymes lipolytiques intéressantes en termes de sélectivité. Pour ces différentes raisons, le potentiel de cette microalgue pour la production de lipides d’intérêt et d’enzymes lipolytiques a été étudié. L’analyse des lipides d’I. galbana a tout d’abord révélé que le DHA était majoritairement greffé sur la position sn-2 des phospholipides. Dans ce contexte, les phospholipides d’I. galbana constituent une matière première intéressante pour la production, par voie enzymatique, de 1-lyso-2–DHA phospholipides, des composés intéressants pour optimiser le transport du DHA au niveau du cerveau. À partir des lipides d’I. galbana, on peut également envisager la production, toujours par voie enzymatique, de phospholipides structurés contenant du DHA et un acide gras à chaîne moyenne ce qui permet de combiner des intérêts thérapeutiques et biologiques intéressants. À partir des ARN totaux d’I. galbana, des séquences codant des enzymes lipolytiques putatives ont été obtenues par RACE et Nested PCR. L’expression hétérologue d’une séquence nommée IgTeCe a été initiée. Une construction plasmidique contenant la séquence codante a été clonée et exprimée avec E. coli. Les résultats ont montré que la * Correspondence: [email protected] This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. F. Hubert et al.: OCL 2017, 24(4), D407 construction plasmidique permettait bien d’obtenir une protéine recombinante avec la masse moléculaire attendue. D’autre part, l’outil d’alignement local de séquences, BLASTP, ainsi que des données biochimiques ont permis de confirmer l’hypothèse que la protéine obtenue était une thioestérase. Mots clés : microalgues / acide gras polyinsaturés v-3 / phospholipides / enzymes lipolytiques 1 Introduction (A) (B) Microalgae are unicellular photosynthetic and eukaryotic Empire : Eukaryota organisms with a wide range of morphologies, shapes, sizes and Kingdom : Chromista aspects. Actually, the total number of algae species has been Phylum : Haptophyta recently estimated to 72,500 despite some uncertainties Class : Coccolithophyceae regarding what organisms should be included as algae or Subclass : Prymnesiophyceae Order : Isochrysidales microalgae (Guiry, 2012). Primary producers of the aquatic Family : Isochrysidaceae ecosystems, microalgae belong, along with the cyanobacteria, to the so-called “phytoplancton” consumed by zooplankton, fish larvae and clams. According to favorable conditions, microalgae Fig. 1. (A) Elements of classification; (B) photograph of Isochrysis have periods of fast growth called “bloom” that can cause red galbana (photonic microscopy Â400). tides. Besides, these tides can sometimes be associated with the production of phycotoxins and lead to harmfull algal blooms “ ” fi a reduced haptonema said vestigial inserted between the (HAB) and shell sh poisoning (Masó and Garcés, 2006). two flagella (Robert et al., 2004). Boosted by the attracting market of biofuels, microalgae Up to date, two strains of Isochrysis were mainly described have received increasing interest during the last decade and and studied in literature: T-Iso and Parke. today they are considered as sustainable natural factories for Isochrysis affinis galbana, more simply designated as the production of a wide range of molecules with potential fi fi T-iso, was formerly said to be a Tahitian clone conspeci c applications in the eld of feed, food, cosmetics, pharmaceu- with I. galbana Parke which originate from temperate zones. tics and energy (Mimouni et al., 2012; Person, 2010). In link with its origin, T-Iso is capable of adapting to a As photosynthetic organisms, microalgae are a source of b much broader range of temperature and light conditions when pigments such as -carotene, that can be found for instance comparing with I. galbana Parke (Ewart and Pruder, 1981; in the halophilic strain Dunaliella salina cultivated on a large Tzovenis et al., 2003). scale along the California coast. Other species like Porphyri- Apart from its plastid coloration which is more yellow, T-Iso dium cruentum are also known to be sources of interesting is similar to Parke from a morphological point of view and no polysaccharides. Nevertheless, the most promising biochemi- difference can be noticed nor by photonic microscopy nor by cal compounds that can be found in microalgae are certainly v scanning electronic microscopy (SEM) (Robert et al., 2004). the lipids for their -3 fatty acids (Mimouni et al., 2015; In 2013, using, as molecular phylogenetic markers, thegenes Poisson et al., 2002) and mainly for the production of biodiesel coding RNA small ribosomal subunit (SSU rRNA), Bendif et al. which has been an important topic for several years now (2013) concluded that the genetic distance between T-Iso and (Cadoret and Bernard, 2008). I. galbana is sufficient to raise T-Iso as a new genius. This new However, according to many literature data, to be interest- genius was called Tisochrysis lutea in reference to the former ing from an economical point of view, biodiesel needs to be name of the strain and to the yellow color of the cells. coproduced with other high added value molecules leading to “ fi ” “ ” In the same area, Pseudoisochrysis paradoxa was also the concepts of bio-re nery and cellular milking (Vinayak renamed Isochrysis galbana. et al., 2015). Before this recent revision, the strains of Isochrysis were In this frame, the present paper is aimed at presenting the not always fully characterized and some confusion may have interest of the microalga Isochrysis galbana for the production occurred in literature as already pointed, more generally for of lipids and lipolytic enzymes with potential applications. whole microalgae, by Muller-Feuga (1997). I. galbana and T. lutea can be obtained worldwide in fi 2 Isochrysis galbana: an overview several culture collections under different speci c strain codes (Tab. 1). This can also be a source of error in literature. To be suitable in mollusc hatchery, algal cells have to Isochrysis galbana is one of the species of the present four main features: Isochrysidales order, belonging to the haptophyta division – a size below